Vibrio vulnificus (V. vulnificus) is an estuarine bacterium that has been associated with severe wound infections or septicemia, particularly in immunocompromised individuals and in persons with conditions such as cirrhosis or hemochromatosis (Blake et al, New Engl. J. Med., 300:1-5 (1979); and Klontz et al, Ann. Intern. Med., 109:318-323 (1980)). Over 50% of persons with septicemia die, and one-third present with shock (Klontz et al, Ann. Intern. Med., 109:318-323 (1980)). The mortality rate among patients who are hypotensive within 24 hours of hospital admission exceeds 90% ((Klontz et al, Ann. Intern. Med., 109:318-323 (1980)). Three-quarters of the patients with septicemia have characteristic bullous skin lesions ((Blake et al, New Engl. J. Med. , 300:1-5 (1979) ; and Klontz et al, Ann. Intern. Med., 109:318-323 (1980)) with histological findings compatible with a toxin-medicated process (Pollack et al, Arch. Intern. Med., 143:837-838 (1983)).
A cytolysin that is lytic for both erythrocytes and Chinese hamster ovary (CHO) cells has been isolated from culture supernatants (Kreger and Lockwood, Infect. Immun., 33:583-590 (1981)). The purified protein has an estimated molecular mass of 56 kilodaltons (kDa). Although V. vulnificus produces both a phospholipase A.sub.2 and a lysophospholipase, these enzymes have been shown to be physically separable from the cytolysin by gel filtration (Testa et al, Infect. Immun., 45:458-463 (1984)). The toxin may bind cholesterol and has been shown to induce the release of K.sup.+ ions, and to a lesser extent Na.sup.+ ions, from liposomes (Yamanka et al, FEMS Microbial. Lett., 44:253-258 (1987)). Other investigators have reported a possible second hemolysin with an estimated molecular mass of 36 kDA, whose hemolytic activity is neutralized by a monoclonal antibody that binds a 36-kDA protein, but not a 56-kDA protein in culture supernatants (Okada et al, J. Gen. Microbial. , 133:2853-2857 (1987)). However, the biological properties of both of these proteins appear to be identical, and the relationship between them is unclear.
Previously, the putative gene for V. vulnificus cytolysin were subcloned on a 3,4-kilobase (kb) insert in pBR325 and designated pCVD702 (Wright et al, Infect. Immun., 50:922-924 (1985)). Both hemolytic and CHO cell-cytotoxic activities were expressed in Escherichia coli, and these activities were neutralized by antisera to the purified 56-kDa cytolysin. This DNA was shown to be specific for V. vulnificus and did not hybridize with DNA from other Vibrio or non-Vibrio species under stringent conditions.
However, homology was demonstrated for all clinical and environmental isolates of V. vulnificus examined, including pathogenic strains (Morris et al, Appl. Environ. Microbial., 53:193-195 (1987)).
To date, practically and commercially suitable nucleic acid probes, specific for pathogenic strains of V. vulnificus, are not known in the art and there has been a need to provide a means to detect and/or diagnose humans or animals infected with pathogenic strains of V. vulnificus. The above-mentioned 3.4-kb subcloned fragment is not suitable for practical or commercial use since testing using this probes requires the use of a sophisticated molecular genetics laboratory, due the need to isolate this relatively large (3.4 kb) fragment from a plasmid immediately prior to each test. Additionally, such a probe requires labeling prior to each test, since such probes cannot be stored for extended periods of time due to loss of labeling.
Accordingly, there is a need to provide an nucleic acid probe specific for pathogenic strains of V. vulnificus which: can be suitably used without the need for a sophisticated molecular genetics laboratory; can be made inexpensively; does not require the use of labeling immediately prior to each test or require the use of radioactive probes; and can be labeled with non-radioactive probes.